Biofilms are three dimensional microbial growth forms comprising bacterial communities and the extracellular matrix they produce. Biofilms are ubiquitous in the environment and may form on solid surfaces where water is available or in suspension, for example as flocs or granules. Biofilms cause significant industrial damage, causing, for example, fouling and corrosion in fluid processes such as water distribution and treatment systems, pulp and paper manufacturing systems, heat exchange systems and cooling towers, and contributing to the souring of oil in pipelines and reservoirs. From a public health perspective, biofilms are also important reservoirs of pathogens in water systems such as drinking water, reservoirs and pipes. Biofilms are also associated with a number of chronic infections in humans, for example otitis media (biofilms on surfaces of the ear), bacterial endocarditis (biofilms on surfaces of the heart and heart valves), cystic fibrosis (biofilms on surfaces of the lungs) and kidney stones, and readily form on medical equipment such as implantable medical devices.
However notwithstanding the significant detrimental effects of biofilms in many environments, biofilms may also be of benefit. For example, in waste water treatment systems suspended floc biofilms or surface-associated biofilms on membranes are said to facilitate nutrient removal, such as in denitrification.
Accordingly, there is a clear need both for effective strategies to eliminate deleterious biofilms and to enhance the activity of beneficial biofilms.
Biofilms are essentially multicellular microbial communities, the formation and development of which is dependent on various multicellular traits of the member organisms, such as cell-cell signalling. Extracellular signalling systems such as quorum sensing are used by bacteria to assess cell density and initiate changes in gene expression and phenotypes when sufficient concentrations of signalling molecules are reached. This is associated with differential gene expression, leading to the induction of, for example, virulence factors and/or defence mechanisms, and with cell differentiation such that biofilm-associated cells become highly differentiated from free-living (planktonic) cells.
As the cells within biofilms differentiate and biofilms mature, reduced metabolic rates, the cellular expression of defence mechanisms and the reduced ability of antimicrobial agents to penetrate the biofilm results in increased antimicrobial resistance and make biofilms particularly difficult to eradicate. Present biofilm control strategies typically target the early stages of biofilm development and involve the use of toxic antimicrobial agents. However such toxic agents present their own downstream problems due to their release into the environment. Improved strategies for biofilm control are clearly required.
It has recently been discovered that Pseudomonas aeruginosa cells within biofilms undergo programmed cell death and lysis in the normal course of the biofilm lifecycle (Webb et al, 2003, Cell death in Pseudomonas aeruginosa biofilm development, J. Bact., 185: 4585-4592). It is believed that programmed cell death in biofilms of P. aeruginosa is prophage-mediated and plays a role in facilitating differentiation and dispersal of a subpopulation of surviving cells from the biofilm.
The present invention is based on the inventors' finding that this phenomenon of programmed cell death in biofilms is linked to the accumulation of reactive oxygen and nitrogen species (RONS) within organisms of the biofilm and that the process of programmed cell death, and dispersal of cells from a biofilm into planktonic cells, can be induced using nitric oxide generators. The ability to increase nitric oxide concentrations in vivo enables the regulation and manipulation of biofilm developmental processes, by promoting programmed cell death, and increases the sensitivity of the cells to antimicrobial agents, thereby providing avenues for inhibiting and/or reversing biofilm development.